Direct effects of canagliflozin on human myocardial redox signalling: a novel role for SGLT1 inhibition

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
H Kondo ◽  
I Akoumianakis ◽  
N Akawi ◽  
C Kotanidis ◽  
A Antonopoulos ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Heart Failure ◽  
Nadph Oxidase ◽  
Heart Rhythm ◽  
Human Myocardium ◽  
Funding Source ◽  
Direct Effects ◽  
Redox Signalling ◽  
Myocardial Biopsies ◽  
Oxide Synthase

Abstract Background Recent clinical trials have demonstrated a role for sodium glucose cotransporter 2 (SGLT2) inhibitors in improving cardiovascular outcomes in heart failure patients, but the underlying mechanisms remain unknown. We investigated the direct effects of canagliflozin, a non-selective SGLT1/SGLT2 inhibitor on myocardial redox signalling in humans. Methods Study 1 included 364 patients undergoing cardiac surgery. Human right atrial appendage biopsies, obtained during surgery, were used to quantify the sources of superoxide (O2.-) and the gene expression of inflammation, fibrosis and myocardial stretch markers. In Study 2, myocardial biopsies from 51 patients were used ex vivo to study the direct effects of canagliflozin on O2.- generation and understand its role in controlling the activity of NADPH-oxidases and uncoupled nitric oxide synthase (NOS). Finally, we used differentiated H9C2 and human primary cardiomyocytes (hCM) to further characterise the key regulatory mechanisms (Study 3). Results SGLT1 was abundantly expressed in the human myocardial biopsies and hCM whilst SGLT2 was barely detectable. SGLT1 expression levels were positively correlated with basal O2.- production and the expression of natriuretic peptides, proinflammatory cytokines and pro-fibrotic markers in human myocardial biopsies from study 1. Incubation of human myocardium with canagliflozin significantly reduced basal and NADPH-oxidase-derived O2.- via AMP kinase (AMPK)-mediated suppression of GTP-activation and consequent reduction of membrane translocation of Rac1, an NADPH-oxidase subunit. This resulted in reduced oxidation and increased bioavailability of tetrahydrobiopterin, the nitric oxide synthase (NOS) co-factor essential for enzymatic coupling, leading to improved NOS coupling. These findings were replicated in hCM, where canagliflozin was shown to regulate AMP/ATP ratio, which could be upstream of AMPK activation. The effects of canagliflozin were significantly attenuated by knocking-down SGLT1 in hCM. Transcriptional profiling of hCM treated with canagliflozin revealed that canagliflozin had striking effects on myocardial redox signalling, causing suppression of apoptotic and inflammatory pathways in the human heart. Conclusions We demonstrate for the first time in humans that canagliflozin suppresses myocardial NADPH-oxidase activity and improves NOS coupling through an SGLT1/AMPK/Rac1-mediated pathway, leading to global anti-inflammatory and anti-apoptotic effects in the human myocardium. These findings provide a mechanistic basis for the beneficial effects of SGLT1/2 inhibitors in patients with heart failure. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): 1. British Heart Foundation (FS/16/15/32047 and PG/13/56/30383 to CA, CH/16/1/32013 to KC, and Centre of Research Excellence award RG/13/1/30181), 2. The Japanese Heart Rhythm Society-European Heart Rhythm Association fellowship grant sponsored by Biotronik.

Localization of endothelial nitric oxide synthase in the normal and failing human atrial myocardium

1996 ◽  
Vol 54 ◽  
pp. 786-787
Author(s):  
Chi-Ming Wei ◽  
Margarita Bracamonte ◽  
Shi-Wen Jiang ◽  
Richard C. Daly ◽  
Christopher G.A. McGregor ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Heart Failure ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Cardiac Tissues ◽  
Mammalian Tissues ◽  
End Stage Heart Failure ◽  
End Stage ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Nitric oxide (NO) is a potent endothelium-derived relaxing factor which also may modulate cardiomyocyte inotropism and growth via increasing cGMP. While endothelial nitric oxide synthase (eNOS) isoforms have been detected in non-human mammalian tissues, expression and localization of eNOS in the normal and failing human myocardium are poorly defined. Therefore, the present study was designed to investigate eNOS in human cardiac tissues in the presence and absence of congestive heart failure (CHF).Normal and failing atrial tissue were obtained from six cardiac donors and six end-stage heart failure patients undergoing primary cardiac transplantation. ENOS protein expression and localization was investigated utilizing Western blot analysis and immunohistochemical staining with the polyclonal rabbit antibody to eNOS (Transduction Laboratories, Lexington, Kentucky).

Abstract 085: CHIP: E3 Ubiquitin Ligase Mediates Proteasomal Degradation of Neuronal Nitric Oxide Synthase in the Paraventricular Nucleus of Rats with Heart Failure

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Neeru M Sharma ◽  
Kenichi Katsurada ◽  
Xuefei Liu ◽  
Kaushik P Patel
Keyword(s):  
Nitric Oxide ◽  
Heart Failure ◽  
Nitric Oxide Synthase ◽  
Paraventricular Nucleus ◽  
Ubiquitin Ligase ◽  
Protein A ◽  
Neuronal Nitric Oxide Synthase ◽  
Proteasomal Degradation ◽  
Oxide Synthase

The exaggerated sympathetic drive is a characteristic of heart failure (HF) due to reduced neuronal nitric oxide synthase (nNOS) within the paraventricular nucleus (PVN). Previously we have shown that there were increased accumulation of nNOS-ubiquitin (nNOS-Ub) conjugates in the PVN of rats with HF (1.0±0.05 Sham vs. 1.29±0.06 HF) due to the increased levels of PIN (a protein inhibitor of nNOS, known to dissociate nNOS dimers into monomers) (0.76±0.10 Sham vs. 1.12±0.09 HF) and decreased levels of tetrahydrobiopterin (BH4): a cofactor required for stabilization of nNOS dimers (0.62±0.02 Sham vs. 0.44±0.03 HF). We also showed that there is blunted nitric oxide-mediated inhibition of sympathetic tone via the PVN in HF. Here we examined whether CHIP(C-terminus of Hsp70 -interacting protein), a chaperone-dependent E3 ubiquitin-protein isopeptide ligase known to ubiquitylate Hsp90-chaperoned proteins could act as an ubiquitin ligase for nNOS in the PVN. Immunofluorescence studies revealed colocalization of nNOS and CHIP in the PVN indicating their possible interaction. CHIP expression was increased by 50% in the PVN of rats with HF(0.96±0.08 Sham vs.1.44±0.10* HF). It is shown that Hsp90 protects nNOS from ubiquitination while Hsp70 promotes the ubiquitination and degradation. We observed significant upregulation of Hsp70 (0.49±0.03 Sham vs. 0.65±0.02* HF) with a trend toward the decrease in Hsp90 expression (0.90±0.07 Sham vs. 0.71±0.06 HF). The opposing effects of the two chaperones could account for the increased CHIP-mediated ubiquitination and degradation of dysfunctional nNOS monomers in the PVN of rats with HF. Furthermore, neuronal NG108-15 cell line transfected with the pCMV3-CHIP-GFP spark (CHIP overexpression plasmid) showed approximately 74% increase in CHIP with concomitant 49% decrease in nNOS expression. In vitro ubiquitination assay in NG108 cells transfected with pCMV-(HA-Ub) 8 and pCMV3-CHIP-GFP spark plasmid reveal increased HA-Ub-nNOS conjugates (1.13 ± 0.09 Scramble vs. 1.65 ± 0.12* CHIP plasmid). Taken together, our results identify CHIP as an E3 ligase for ubiquitination of dysfunctional nNOS and CHIP expression is augmented during HF leading to increased proteasomal degradation of nNOS in the PVN.

Abstract 103: ACE2 Overexpression Prevents DOCA-Salt Hypertension by Modulating Oxidative Stress and Nitric Oxide in the Central Nervous System

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Srinivas Sriramula ◽  
Huijing Xia ◽  
Eric Lazartigues
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Central Nervous System ◽  
Nervous System ◽  
Nadph Oxidase ◽  
Salt Treatment ◽  
Salt Hypertension ◽  
The Central Nervous System ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Elevated reactive oxygen species (ROS) in the central nervous system (CNS) through NADPH oxidase and diminished Nitric oxide (NO) levels are involved in the pathogenesis of hypertension. We previously reported that central Angiotensin Converting Enzyme 2 (ACE2) overexpression prevents the development of hypertension induced by DOCA-salt in a transgenic mouse model (syn-hACE2; SA) with human ACE2 targeted selectively to neurons in the CNS. While baseline blood pressure (BP; telemetry) was not different among genotypes, DOCA-salt treatment (1mg/g body wt DOCA, 1% saline in drinking water for 3 weeks) resulted in significantly lower BP level in SA mice (122 ±3 mmHg, n=12) compared to non-transgenic (NT) littermates (138 ±3 mmHg, n=8). To elucidate the mechanisms involved in this response, we investigated the paraventricular nucleus (PVN) expression of Nox-2 (catalytic subunit of NADPH oxidase), 3-nitrotyrosine, and endothelial nitric oxide synthase (eNOS) and anti-oxidant enzymes superoxide dismutase (SOD) and catalase in the hypothalamus. DOCA-salt treatment resulted in decreased catalase (95.2 ±5.6 vs. 113.8 ±17.6 mmol/min/ml, p<0.05) and SOD (4.1 ±0.4 vs. 5.9 ±0.2 U/ml, p<0.01) activities in hypothalamic homogenates of NT mice, which was prevented by ACE2 overexpression (141.8 ±9.9 vs. 142.1 ±9.2 mmol/min/ml and 5.9 ±0.3 vs. 7.9 ±0.2 U/ml, respectively). NT mice treated with DOCA-salt showed increased oxidative stress as indicated by increased expression of Nox-2 (61 ±5 % increase, n=9, p<0.001 vs. NT) and 3-nitrotyrosine (89 ±32 % increase, n=9, p<0.01 vs. NT) in the PVN which was attenuated in SA mice. Furthermore, DOCA-salt hypertension resulted in decreased phosphorylation of eNOS-ser1177 in the PVN (33 ±5 % decrease, n=9, p<0.05 vs NT) and this decrease was prevented by ACE2 overexpression. Taken together, these data provide evidence that brain ACE2 regulates the balance between NO and ROS levels, thereby preventing the development of DOCA-salt hypertension.

Abstract 15295: Molecular Mechanism of Chronic Sildenafil-Caused Regression of Heart Failure: Effects on the Express of Cardiac SR Ca2+-ATPase, Subtype of β-Adrenergic Receptors and Nitric Oxide Synthase Isoforms

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Heng-Jie Cheng ◽  
Tiankai Li ◽  
Che Ping Cheng
Keyword(s):  
Nitric Oxide ◽  
Heart Failure ◽  
Nitric Oxide Synthase ◽  
Adrenergic Receptors ◽  
Left Ventricular ◽  
Male Rats ◽  
Protein Levels ◽  
Myocyte Function ◽  
Oxide Synthase ◽  
Β Adrenergic Receptors

Background: Sildenafil (SIL), a selective inhibitor of PDE5 has been shown to exert profound beneficial effects in heart failure (HF). Recently we further found that SIL caused regression of cardiac dysfunction in a rat model with isoproterenol (ISO)-induced progressive HF. However, the molecular basis is unclear. We hypothesized that reversal of HF-induced detrimental alterations on the expressions of cardiac SR Ca 2+ -ATPase (SERCA2a), β-adrenergic receptors (AR) and nitric oxide synthase (NOS) isoforms by SIL may play a key role for its salutary role in HF. Methods: Left ventricular (LV) and myocyte function and the protein levels of myocyte β 1 - and β 3 - AR, SERCA2a, phospholamban (PLB) and three NOS were simultaneously evaluated in 3 groups of male rats (6/group): HF , 3 months (M) after receiving ISO (170 mg/kg sq for 2 days); HF/SIL , 2 M after receiving ISO, SIL (70 μg/kg/day sq via mini pump) was initiated and given for 1 M; and Controls (C). Results: Compared with controls, ISO-treated rats progressed to severe HF at 3 M after ISO followed by significantly decreased LV contractility (E ES , HF: 0.7 vs C: 1.2 mmHg/μl) and slowed LV relaxation, reductions in the peak velocity of myocyte shortening (77 vs 136 μm/sec), relengthening (62 vs 104 μm/sec) and [Ca 2+ ] iT (0.15 vs 0.24) accompanied by a diminished myocyte inotropic response to β-AR agonist, ISO (10 -8 M). These abnormalities were associated with concomitant significant decreases in myocyte protein levels of β 1 -AR (0.23 vs 0.64), SERCA2a (0.46 vs 0.80), PLB Ser16 /PLB ratio (0.24 vs 0.40) and eNOS (0.28 vs 0.46), but significantly increases in protein levels of β 3 -AR (0.29 vs 0.10) and iNOS (0.18 vs 0.08) with relatively unchanged nNOS. Chronic SIL prevented the HF-induced decreases in LV and myocyte contraction, relaxation, peak [Ca 2+ ] iT , and restored normal myocyte contractile response to ISO stimulation. With SIL, protein levels of myocyte β 1 - and β 3 -AR, SERCA2a were restored close to control values, but eNOS was significantly elevated than controls (0.77). Conclusions: Chronic SIL prevents HF-caused downregulation of cardiac β 1 -AR and reverse contrast changes between iNOS and β 3 -AR with SERCA 2a and eNOS expression, leading to the preservation of LV and myocyte function, [Ca 2+ ] iT , and β-adrenergic reserve.

Abstract 15532: Altered Ubiquitination and Stability of Protein Inhibitor of Neuronal Nitric Oxide Synthase in the Paraventricular Nucleus of Chronic Heart Failure Rats: Role of Angiotensin II

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Neeru Sharma ◽  
Xuefei Liu ◽  
Hong Zheng ◽  
Kaushik Patel
Keyword(s):  
Nitric Oxide ◽  
Heart Failure ◽  
Angiotensin Ii ◽  
Chronic Heart Failure ◽  
Paraventricular Nucleus ◽  
Neuronal Nitric Oxide Synthase ◽  
Ang Ii ◽  
Protein Inhibitor ◽  
Ubiquitin Proteasome ◽  
Oxide Synthase

Introduction and Hypothesis: Expression of neuronal nitric oxide synthase (nNOS) is decreased in the paraventricular nucleus (PVN) of rats with chronic heart failure (CHF), however the underlying molecular mechanism/s remain unclear. Recently, we demonstrated, Angiotensin II (Ang II) mediated increase in PIN: protein inhibitor of nNOS (0.76±0.10 Sham vs 1.12±0.09* CHF) which is known to down-regulate nNOS through disruption of active dimers (~60% decrease in dimer/monomer ratio) in the PVN of rats with CHF. Functionally impeded monomeric enzyme is degraded by ubiquitin proteasome system. Interestingly, PIN transcript levels remain unchanged in the PVN in CHF (1.00±0.23 Sham vs. 1.1±0.28 CHF). This observation prompted us to elucidate the molecular mechanism for the accumulation of PIN post-transcriptionally in the PVN in CHF Methods and Results: We used coronary artery ligation model of CHF in rats (6-8 weeks past ligation) and neuronal NG108-15 hybrid cell line. PIN translation was inhibited using cyclohexamide (CHX) for 0-4h after 20h of pretreatment with Ang II in NG108 cells. CHX mediated decrease in PIN expression was ameliorated with Ang II (0.19±0.04 vs 0.41±0.06* 4h). Proteasome inhibitor lactacystin (LC) treatment dramatically elevates PIN level suggesting the involvement of proteasome system in PIN regulation. Immunoprecipitation with ubiquitin antibody showed decrease PIN-Ub conjugates in Ang II-treated cells (1.04 ± 0.05 LC vs. 0.62 ± 0.07* LC AngII). In vitro ubiquitination assay in cells transfected with pCMV-(HA-Ub)8 vector revealed reduction of HA-Ub-PIN conjugates after Ang II treatment (9.2 ± 2.2 LC vs. 4.5 ± 0.6* LC Ang II). Furthermore, there was decreased accumulation of PIN-Ub conjugates in the PVN of CHF rats compared to Sham as revealed by immunohistochemistry. Conclusions: Taken together, our studies revealed that PIN is targeted for rapid degradation by the ubiquitin-proteasome pathway and Ang II delays the rate of degradation resulting in accumulation of PIN. We conclude that post-translational accumulation of PIN, mediated by Ang II, leads to a decrease in the dimeric active form of nNOS as well as protein levels of nNOS, which may lead to reduced nitric oxide resulting in over-activation of sympathetic drive during CHF.

Abstract P388: Extra-cardiac BCAA Catabolism Lowers Blood Pressure And Protects From Heart Failure

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Danielle Murashige ◽  
Cholsoon Jang ◽  
Michael Neinast ◽  
Michael Levin ◽  
Jae Woo Jung ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Heart Failure ◽  
Blood Pressure ◽  
Mendelian Randomization ◽  
Multiple Models ◽  
Direct Effects ◽  
Heavy Isotope ◽  
Branched Chain Amino Acid ◽  
Branched Chain

Pharmacologic activation of branched chain amino acid (BCAA) catabolism is protective in numerous models of heart failure (HF). How this protection occurs has remained unclear, although a causative block in cardiac BCAA oxidation has been proposed. We use here in vivo heavy isotope infusion studies to show that cardiac preference for BCAA oxidation increases, rather than decreases, in multiple models of HF. We use various genetic models to show that cardiac-specific activation of BCAA oxidation does not protect from HF, even though systemic activation of BCAA oxidation does. Lowering plasma and cardiac BCAAs by genetic means is also not sufficient to confer protection comparable to that conferred by pharmacologic activation of BCAA oxidation, suggesting alternative mechanisms of protection. Surprisingly, telemetry and invasive hemodynamic studies showed that pharmacological activation of BCAA catabolism lowers blood pressure, a well-established cardioprotective mechanism. The effects on blood pressure occurred independently of nitric oxide (NO), and reflected a vascular resistance to adrenergic constriction. Finally, mendelian randomization studies revealed that elevations in plasma BCAAs portend higher blood pressure in large human cohorts. Together, these data indicate that activation of BCAA oxidation lowers blood pressure and protects from heart failure independently of any direct effects on the heart itself.

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